Articular cartilage, the avascular tissue that covers the ends of synovial joints and provides articulating joints with a durable, weight-distributing surface, damaged by illness or trauma has little capacity for self-repair. Current interventions that include autologous or heterologous transplantation, articular resurfacing, are of limited efficacy, and in the long term, untreated lesions may result in large-scale degenerative changes and osteoarthritis. Hence bioreactor and bioprocess strategies to generate tissue-engineered articular cartilage continue to be researched, as replacement of diseased or damaged cartilage represents an important medical problem that remains unsolved. The long-term goal of our research is to design and develop bioreactors or bioprocessing units that can be used to generate engineered-tissues. An important component of our strategy to generate tissue-engineered constructs is the use of bioreactors to provide adequate stimulus to the cell-seeded scaffolds. Our objective in this R21 grant application is to develop a bioreactor that utilizes stimulation by ultrasound, and conduct a detailed study on the effect of ultrasound on chondrocytes cultured in 3-D scaffolds in vitro. We will achieve the objective of this application by pursuing the following specific aims: Specific aim 1: To develop a bioreactor that uses continuous ultrasound to enhance cartilage matrix formation and maintain chondrocyte differentiation. Specific Aim 2: To implement a feedback loop into the US-based bioreactor. Specific Aim 3: To evaluate the effects of continuous ultrasound on the morphology and cytoskeleton of chondrocytes maintained in the bioreactors under development. Public Health Relevance Statement: At the conclusion of the R21 phase of the proceed research, we expect to have designed and developed a novel US-aided bioreactor configuration that incorporates a feed-back control to provide different regimes of mechanical conditioning to engineered tissues at different stages of development. We propose that the novelty of this strategy lies with the assessment of the cellular response to ultrasound exposure and, the application of this technology to efficiently and effectively grow cells (i.e. chondrocytes) in 3D culture systems. While bioreactors based on rotation and compression are well described, much remains to be learned about bioreactors based on US stimulation. In summary, while US has been shown to impact cartilage function at the cellular level, there is still a need to better understand the effect of US stimulation of chondrocytes seeded and maintained in 3-D scaffolds, which are better representatives of chondrocytes in-vitro culture. At the completion of this research, we expect to: 1) better understand how mechanical stimulation by ultrasound influences cell activity in 3-D scaffolds and 2) better understand the complex interplay between various factors (mechanical and biochemical cues) influencing tissue formation. Finally, we expect to have developed a highly reproducible in vitro model system of chondrocyte 3D-culture that likely uses US, which will allow us to carry out a detailed examination of the mechanisms of signal transduction processes in a future R-01 application.